Tumorigenesis and/or proliferation involve interactions of neoplastic cells with each other and with non-malignant host cells. Binding of diffusible factors with cognate receptors mediate many of these interactions. Bombesin-like peptides (BLPs) are a family of regulatory neuropeptides. Gastrin-releasing peptide (GRP) is a human BLP that elicits many physiological effects including mitogenesis. GRP induces cell proliferation by binding to a GRP-preferring G-protein coupled receptor (GRPr) and activating several signal transduction cascades. GRPr and other G protein coupled receptors (GPCRs) may be involved in the promotion and proliferation of several malignancies. Therefore, interruption of GRP-dependent signaling offers a potentially powerful approach for treating cancers expressing GRPr. Elucidating molecular pathways involved in GRPr-mediated signaling and growth may uncover additional targets for chemotherapy, or strategies to combine anti-GPCR therapy with other agents. Therefore, we are studying the function and regulation of GRPr. One domain that is likely to be crucial for some aspects of GRPr-mediated signaling is the carboxyl terminal domain (CTD). CTDs of many GPCRs including GRPr contain sites for phosphorylation, and CTD are involved in regulatory processes including internalization and desensitization. Recent data has implicated the CTD of some GPCRs in activation of signaling. Thus, the CTD of GRPr is likely to be required for regulation of GRP signaling. A central goal of this application is to determine how the CTD of GRPr and GRPr phosphorylation affect receptor-mediated signaling and desensitization. Several complementary approaches will be used: (1) functions and regulation of GRPr mutants which are poor substrates for agonist-induced phosphorylation will be studied in a transfected cell system and compared with wildtype GRPr, (2) GRPr phosphorylation sites will be determined using mass spectrometry, and (3) Novel signaling partners for GRPr will be identified and characterized. Candidate GRPr binding proteins have been identified in a yeast two-hybrid screen in which the CTD was used as a bait. The candidate signaling partners will be evaluated using assays that (a) document and analyze GRPr-binding protein interactions, and (b) determine the roles of GRPr binding proteins in signaling in intact cells. Tumor cells expressing endogenous GRPr will be studied to assess the physiological relevance of the results from each Specific Aim. [unreadable] [unreadable]